A quantitative enzyme-linked immunoassay (elisa) to approximate complement fixing antibody titers in serum from patients with coccidioidomycosis

ABSTRACT

Coccidioidomycosis is most often diagnosed serologically and the quantitative complement-fixing antibody test (CF) is considered prognostically useful. Because CF is complex, labor-intensive, and poorly standardized, an enzyme-linked immunoassay (ELISA) alternative would be attractive. The present invention features an antibody-binding domain that is restricted to a 200 amino acid recombinant peptide of the known antigen responsible for CF activity. Overlapping truncations of this peptide do not bind CF antibodies, suggesting that the responsible epitope(s) are conformational. Further, anchoring the antigenic peptide to the ELISA plate by means of a C-terminal tag instead of allowing the peptide to randomly adhere to the plastic plate improves sensitivity of antibody detection by one to two logs in different sera. The newly developed ELISA shows a significant quantitative correlation with CF. This ELISA shows potential as the basis for a new quantitative assay for coccidioidal antibodies.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part and claims benefit of U.S.patent application Ser. No. 16/058,538 filed Aug. 8, 2018, which is anon-provisional and claims benefit of U.S. Patent Application 62/542,594filed Aug. 8, 2017, the specification(s) of which is/are incorporatedherein in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. R01Al132140 awarded by National Institutes of Health. The government hascertain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

Applicant asserts that the paper copy of the Sequence Listing isidentical to the Sequence Listing in computer readable form found on theaccompanying computer file, entitledUNIA_17_33_CIP_Sequence_Listing_ST25. The content of the sequencelisting is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention features a recombinant Cts1 peptide and method toutilize the recombinant Cts1 peptide in an enzymes-linked immunoassay(ELISA) for the detection of a coccidioidomycosis infection.

BACKGROUND OF THE INVENTION

Coccidioidomycosis (CM), also known as San Joaquin Valley fever, is botha regionally and nationally important systemic fungal infection, oftenconfused with other respiratory infections, cancer, or rheumatologicconditions. Because the clinical manifestations of CM overlap with sucha diverse range of other illnesses, diagnosis typically requiresspecific laboratory testing. While direct microscopic detection ofspherules in clinical specimens, growth of the fungus in culture, PCRdetection of Coccidioides-specific of DNA sequence, or measurement ofcoccidioidal antigen are all clinically available approaches, diagnosisof CM is most frequently accomplished by detecting specificanti-coccidioidal antibodies in serum. Of the several serologic testscommercially performed for this purpose, the assay for anti-coccidioidalcomplement-fixing (CF) antibodies and the immunodiffusion methoddesigned to measure the antibodies directed against the same antigen arethe only tests whose quantitation has prognostic value, and for thisreason they have remained in clinical use for over the past 60 years.

Despite its long history, the CF antibody assay has several limitationsfor all but the most specialized laboratories. Several biologicsreagents (tanned sheep red blood cells, fresh serum complement,heat-sensitive coccidioidal antigen preparations) are required for theprocedure, each of which can be obtained from diverse suppliers. Thetest is complex and labor-intensive. Day-to-day agreement amongreplicates is not always achieved and for this reason some laboratoriesrepeat a prior specimen contemporaneously with a current one todetermine more directly any difference in titer between the two.Recommended procedures are not uniform, there is no informationavailable regarding differences obtained between different referencelaboratories, and a national performance testing program has not beenestablished. A method which avoided these limitations would be a usefulalternative.

There is broad agreement that the protein within complex coccidioidalextracts that reacts with CF antibodies is the 427 amino acid product ofthe chitinase gene, CTS1, and its crystal structure has been determined.The recombinant expression product of CTS1 (rCTS1) has been consideredas a reagent for an enzyme-linked immunoassay (ELISA) to measure CFantibodies, found to have potential, but was not pursued further.Truncations rCTS1₂₀₋₁₁₁ and rCTS1₂₈₀₋₄₂₇ were not reactive with serafrom patients with CM. Little has been done further with theseobservations reported over twenty years ago, perhaps in large partbecause coccidioidomycosis is an orphan disease for which relativelysmall commercial incentive exists to improve its diagnosis andmanagement.

Described herein, is additional work with recombinant products oftruncations of rCTS1. The domain immunoreactive with antibodies isfurther restricted in serum from patients with CM and characterized theepitope(s) responsible for antibody binding. Also, assay results withreference to a standard curve, permitting expression of antibody bindingas an antibody concentration instead of titers. These results support arecombinant truncation of CTS1 as a useful reagent for the eventual tranon of quantitative CF antibody assays to an ELISA platform.

BRIEF SUMMARY OF THE INVENTION

It is an objective of the present invention to provide a recombinantCts1 peptide composition and method to utilize the recombinant Cts1peptide in an Enzyme-Linked Immunoassay (ELISA) that allow for thedetection of a coccidioidomycosis infection, as specified in theindependent claims. Embodiments of the invention are given in thedependent claims. Embodiments of the present invention can be freelycombined with each other if they are not mutually exclusive.

The present invention features an isolated Cts1 peptide having sequencethat is at least 90% identical to SEQ ID NO: 4 and containing at leastone substitution modification relative to SEQ ID NO: 4.

Additionally, the present invention may feature an assay platform fordetecting anti-coccidioidal antibodies. In some embodiments the platformcomprises a solid support and an isolated Cts1 peptide having a sequenceaccording to SEQ ID NO: 4 and containing at least one substitutionmodification relative to SEQ ID NO: 4, wherein the Cts1 is peptideattached to the solid support.

The present invention may also feature an isolated Cts1 peptide having asequence that is at least 90% identical to SEQ ID NO: 4, wherein theCts1 peptide is covalently attached to a component, wherein thecomponent attaches to a peptide that is coated onto a solid support.

One of the unique and inventive technical features of the presentinvention is the use of a biotin-mimic tag sequence to orient thebinding of the CTS1 peptide to a streptavidin coated well. Withoutwishing to limit the invention to any theory or mechanism, it isbelieved that the technical feature of the present inventionadvantageously provides for the CTS1 peptide conformation to bemaintained and allows for an increase in sensitivity for the assay. Thistechnical modification improves the quantitative ELISA assay. Ingeneral, ELISA assays are less complex, ELISA reagents are more readilyavailable, the format is more familiar to clinical laboratories.Additionally, ELISAs are less labor intensive and can be automated. Aquantitative ELISA measuring CS Abs to rCTS1₁₀₅₋₃₁₀ may be moresensitive than existing serologic tests for early coccidioidalinfection. Additionally, methods and compositions of the presentinvention help reduce the non-specific antibody binding unrelated toacquiring a coccidioidal infection. This allows for a more sensitivetest for early coccidioidal infection because signals could bedistinguished at lower intensity, avoiding confusion with nonspecificantibody binding. None of the presently known prior references or workhas the unique inventive technical feature of the present invention.

The present invention was able to determine a much smaller specifictruncation of the 427 amino acid product of the chitinase gene, CTS1,that binds all of the highly specific anti-coccidioidal antibodies inserum than had been accomplished in the past. Previous truncations ofthe CTS1 protein showed binding to rCTS1₂₀₋₃₁₀, however, amino acids 20to 309 were presently discovered to have no role in the binding.Additionally, truncations such as rCTS1₂₀₋₁₁₁ and rCTS1₂₈₀₋₄₂₇ were,also, shown to not be reactive with sera from patients with CM. The useof the CTS1 truncation (SEQ ID NO: 4) along with a single mutation helpsto reduce the non-specific antibody binding unrelated to acquiring acoccidioidal infection. Overall, this provides a more sensitive test forearly coccidioidal infection because signals could be distinguished atlower intensity, avoiding confusion with nonspecific antibody binding.Very surprisingly, smaller overlapping truncations from amino acid 110to amino acid 310 showed virtually no specific antibody binding whichunexpectedly indicates that all of the specific antibody binding is tosites dependent on conformation that is retained with SEQ ID NO: 4 butis not present on smaller portions of the truncation.

Furthermore, the prior references teach away from the present invention.For example, standard CF antibody assays are complex and laborintensive, with minimal reproducibility from day-to-day. Additionally,reagents are harder to obtain and may differ depending on the supplierin which it is acquired. Moreover, there are no uniform recommendedprocedures and no established national performance testing program.Furthermore, the inventive technical features of the present inventioncontributed to a surprising result. For example, the antibody binding toCoccidioides-specific epitope(s) depends upon conformation(s) present inthe CTS1 peptide which is lost with the individual smaller peptides.Therefore, binding of serum antibodies to the CTS1 peptide was directedat conformational or discontinuous epitopes rather than epitopes of aprimary amino acid sequence.

Additionally, although prior art has described an antigen enzymeimmuno-assay (EIA) to detect coccidioidomycosis, prior assays were todetect individuals with more severe infections of coccidioidomycosis,described herein is a method to detect early infections. Furthermore,prior EIA assays use urine samples to detect galactomannans, however,the present invention uses serum samples to detect Cts1 protein.Therefore, one could not anticipate that because an EIA assay works witha protein detected in the urine that an EIA assay will be successful fordetecting protein in a serum sample even if the two tests are to detectcoccidioidomycosis infections.

Moreover, the present invention features an isolated Cts1 protein fromthe Coccidioides posadasii species. However, there are two Coccidioidesspecies which can cause infection in humans, C. posadasii and C.immitis. The two species are morphologically identical, but theirpredicted proteins are only about 90% homologous. Therefore, to compareproteins between the species would potentially lead to minor differencesin protein sequences. In the present invention, the SEQ ID NO: 4 isextracted from C. posadasii and has at least one mutation per thatspecies protein.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent application or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

The features and advantages of the present invention will becomeapparent from a consideration of the following detailed descriptionpresented in connection with the accompanying drawings in which:

FIGS. 1A-1B show binding of anti-rCTS1(aa111-310) antibodies torCTS1(aa111-310) after Ni-NTA affinity purification. FIG. 1A shows anSDS-PAGE rCTS1(aa20-310) (34.7 kDa) and rCTS1(aa111-310) (27.4 kDa).FIG. 1B shows an immunoblot of the same peptides with human CF+ sera.The larger bands are presumably dimers.

FIG. 2 shows an ELISA optical densities for IgG antibodies in human CF+sera at different dilutions or PBS alone that bind to rCTS1₁₀₅₋₃₁₀, fouroverlapping peptides spanning rCTS1₁₀₅₋₃₁₀ (Frag1-Frag4), MBP alone, orPBS. The figure shows evidence of no binding for four subunits of Cts1,whereas CTS₁₀₅₋₃₁₀ has complete binding. This may be a prototype for adiagnostic test procedure.

FIG. 3 shows the relationship of anti-rCTS1₁₀₅₋₃₁₀ antibodyconcentrations measured by ELISA to CF antibody titers for 50 individualhuman CF+ sera. The dotted lines represent the 95% confidence intervalsfor the slope of the regression line.

FIG. 4 shows the absorption of CF antibodies with increasingconcentrations of rCTS1₁₁₁₋₃₁₀ (27.4 kDa) eliminates binding of CFantibodies to rCTS1₂₀₋₃₁₀ (34.7 kDa). The blank lane represents loadingcontrol.

FIGS. 5A-5B show the lack of immunoreactivity of rCTS1 fragments 1-4.FIG. 5A shows an SDS PAGE and FIG. 5B shows an immunoblot ofrCTS1₁₀₅₋₃₁₀, 4 subunits of rCTS1₁₀₅₋₃₁₀, and MBP probed with CFantibody-containing human sera. Only subunit #2 is cleaved from MBP.

FIG. 6 shows ELISA and CF activities are comparable. Briefly, microtiterplates were prepared using 100 ng of CTS1 111-310 per well as describedabove. Human sera with CF titers from undetectable to 1:64 were diluted1:100 and then proportionally to the CF titer. For example, sera of CFtiter of 1:1 were diluted 1:100 and sera of CF titer of 1:64 werediluted 1:6400. Blocking prior to sera application, and enzyme conjugatedetection of antibodies and absorbance measurements were conducted asdescribed above.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compounds, compositions, and/or methods are disclosedand described, it is to be understood that this invention is not limitedto specific synthetic methods or to specific compositions, as such may,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

The present invention features methods and compositions for detectingcoccidioidomycosis. Inventors surprisingly found that all or most of theantibody binding (CF antibody binding) is directed toward amino acids105-310 or amino acids 111-310. The methods of the present inventionallow for a specific detection above non-immune sera at a 1:100 dilution(an increase in sensitivity). The methods and compositions of thepresent invention will help reduce the non-specific antibody bindingunrelated to acquiring a coccidioidal infection. This could provide amore sensitive test for early coccidioidal infection because signalscould be distinguished at lower intensity, avoiding confusion withnonspecific antibody binding.

Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Similarly, the word “or” is intended toinclude “and” unless the context clearly indicates otherwise.“Comprising” means “including.” Hence “comprising A or B” means“including A” or “including B” or “including A and B.”

Suitable methods and materials for the practice and/or testing ofembodiments of the disclosure are described below. Such methods andmaterials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein can be used. For example, conventional methods well known in theart to which the disclosure pertains are described in various generaland more specific references, including, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, 1989; Sambrook et al., Molecular Cloning: A LaboratoryManual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates, 1992 (andSupplements to 2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlowand Lane, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1999, the disclosures of which are incorporated intheir entirety herein by reference.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Although methods and materials similar or equivalent to those describedherein can be used to practice or test the disclosed technology,suitable methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Sequence identity: The identity (or similarity) between two or morenucleic acid sequences is expressed in terms of the identity orsimilarity between the sequences. Sequence identity can be measured interms of percentage identity; the higher the percentage, the moreidentical the sequences are. Sequence similarity can be measured interms of percentage similarity (which takes into account conservativeamino acid substitutions); the higher the percentage, the more similarthe sequences are. Methods of alignment of sequences for comparison arewell known in the art. Various programs and alignment algorithms aredescribed in: Smith & Waterman, Adv. Appl. Math, 2:482, 1981; Needleman& Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988;Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al, Nuc. Acids Res,16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al., Meth. Mol. Bic. 24:307-31, 1994.Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailedconsideration of sequence alignment methods and homology calculations.The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J,Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biotechnology (NCBI, National Libraryof Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on theInternet, for use in connection with the sequence analysis programsblastp, blastn, blastx, tblastn and tblastx. Additional information canbe found at the NCBI web site. BLASTN may be used to compare nucleicacid sequences, while BLASTP may be used to compare amino acidsequences. If the two compared sequences share homology, then thedesignated output file will present those regions of homology as alignedsequences. If the two compared sequences do not share homology, then thedesignated output file will not present aligned sequences. TheBLAST-like alignment tool (BLAT) may also be used to compare nucleicacid sequences (Kent, Genome Res. 12:656-664, 2002). BLAT is availablefrom several sources, including Kent Informatics (Santa Cruz, Calif.)and on the Internet (genome.ucsc.edu). Once aligned, the number ofmatches is determined by counting the number of positions where anidentical nucleotide or amino acid residue is presented in bothsequences. The percent sequence identity is determined by dividing thenumber of matches either by the length of the sequence set forth in theidentified sequence, or by an articulated length (such as 100consecutive nucleotides or amino acid residues from a sequence set forthin an identified sequence), followed by multiplying the resulting valueby 100. For example, a nucleic acid sequence that has 1166 matches whenaligned with a test sequence having 1554 nucleotides is 75.0 percentidentical to the test sequence (1166÷1554*100=75.0). The percentsequence identity value is rounded to the nearest tenth.

Sample: Any composition containing or presumed to contain a biomarker,or a composition being tested for the presence or absence of aparticular biomarker or other biological entity such as an antibody orfragment thereof. Samples may include purified or separated componentsof cells, tissues, or blood, e.g., DNA, RNA, proteins, cell-freeportions, or cell lysates. The sample can also be from a fresh liquidsample or a previously frozen sample. In certain embodiments, the sampleis a liquid sample, e.g., blood or a blood component (plasma or serum),urine, semen, saliva, sputum, mucus, semen, tear, lymph, cerebral spinalfluid, material washed from a swab, etc. The sample can also bepartially processed from a sample directly obtained from an individual.

Sensitivity: As used herein sensitivity may refer to the ability of atest to detect antibodies that are specifically produced by a patient'sresponse to a coccidioidal infection. In some embodiments being moresensitive is to mean that a test is able to detect specific andtherefore diagnostic antibodies than another test.

Referring now to FIGS. 1A-6, in some embodiments, the present inventionfeatures the use of a recombinant Cts1 peptide which is utilized in anELISA assay for the detection of a coccidioidomycosis infection. Forreference, the sequence for chitinase (SEQ ID NO: 1; Coccidioidesposadasii, GenBank: AAA92643.1) is shown below in Table 1.

In other embodiments, the present invention features an isolated Cts1peptide having a sequence that is at least 90% identical to SEQ ID NO: 4and containing at least one substitution relative to SEQ ID NO: 4.Non-limiting embodiments of the isolated Cts1 peptides are shown inTable 1.

TABLE 1 Chitinase (Cts1) and isolated Cts1 peptide sequences SEQ ID NO:DESCRIPTION SEQUENCE 1 Chitinase (Cts1) MRFLIGALLT LQTLVQASSM SSMPNYYPVPEAPAEGGFRS VVYFVNWAIY GRGHNPQDLK ADQFTHILYA FANIRPSGEV YLSDTWADTDKHYPGDKWDE PGNNVYGCIK QMYLLKKNNR NLKTLLSIGG WTYSPNFKTP ASTEEGRKKFADTSLKLMKD LGFDGIDIDW EYPEDEKQAN DFVLLLKACR EALDAYSAKH PNGKKFLLTIASPAGPQNYN KLKLAEMDKY LDFWNLMAYD FSGSWDKVSG HMSNVFPSTT KPESTPFSSDKAVKDYIKAG VPANKIVLGM PLYGRAFAST DGIGTSFNGV GGGSWENGVW DYKDMPQQGAQVTELEDIAA SYSYDKNKRY LISYDTVKIA GKKAEYITKN GMGGGMWWES SSDKTGNESLVGTVVNGLGG TGKLEQRENE LSYPESVYDN LKNGMPS 2 Cts1₂₀₋₃₁₀MSSMPNYYPVP EAPAEGGFRS VVYFVNWAIY GRGHNPQDLK ADQFTHILYA FANIRPSGEVYLSDTWADTD KHYPGDKWDE PGNNVYGCIK QMYLLKKNNR NLKTLLSIGG WTYSPNFKTPASTEEGRKKF ADTSLKLMKD LGFDGIDIDW EYPEDEKQAN DFVLLLKACR EALDAYSAKHPNGKKFLLTI ASPAGPQNYN KLKLAEMDKY LDFWNLMAYD FSGSWDKVSG HMSNVFPSTTKPESTPFSSD KAVKDYIKAG VPANKIVLGM PLYGRAFAST DGIGTSFNGV 3 Cts1₁₁₁₋₃₁₀QMYLLKKNNR NLKTLLSIGG WTYSPNFKTP ASTEEGRKKF ADTSLKLMKD LGFDGIDIDWEYPEDEKQAN DFVLLLKACR EALDAYSAKH PNGKKFLLTI ASPAGPQNYN KLKLAEMDKYLDFWNLMAYD FSGSWDKVSG HMSNVFPSTT KPESTPFSSD KAVKDYIKAG VPANKIVLGMPLYGRAFAST DGIGTSFNGV 4 Cts1₁₀₅₋₃₁₀ VYGCIK QMYLLKKNNR NLKTLLSIGGWTYSPNFKTP ASTEEGRKKF ADTSLKLMKD LGFDGIDIDW EYPEDEKQAN DFVLLLKACREALDAYSAKH PNGKKFLLTI ASPAGPQNYN KLKLAEMDKY LDFWNLMAYD FSGSWDKVSGHMSNVFPSTT KPESTPFSSD KAVKDYIKAG VPANKIVLGM PLYGRAFAST DGIGTSFNGV 5Modified Cts1₁₀₅₋₃₁₀ 99% VYGCIK QMYLLKKNNR NLKTLLSIGGidentical to SEQ ID NO: WTYSPNFKTP ASTEEGRKKF ADTSLKLMKD4; bold letters are LGFDGIDIDW EYPEDEKQAN DFVLLLKACRsubstituted amino acids EA V DAYSAKH PNGKKFL V TI ASPAGPQNYNKLKLAEMDKY LDFWNLMAYD FSGSWDKVSG HMSNVFPSTT KPESTPFSSD KAVKDYIKAGVPANKIVLGM PLYGRAFAST DGIGTSFNGV 6 Modified Cts1₁₀₅₋₃₁₀ 99%VYGCIK QMYLLKKNNR NLKTLLSIGG identical to SEQ ID NO:WTYSPNFKTP ASTEEGRKKF  L DTSLKLMKD 4: bold letters are LGFDGIDIDW EYP DDEKQAN DFVLLLKACR substituted amino acidsEALDAYSAKH PNGKKFLLTI ASPAGPQNYN KLKLAEMDKY LDFWNLMAYD FSGSWDKVSGHMSNVFPSTT KPESTPFSSD KAVKDYIKAG VPANKIVLGM PLYGRAFAST DGIGTSFNGV 7Modified Cts1₁₀₅₋₃₁₀ 98% VYGCIK QMYLLKKNNR NLKTLLSIGGidentical to SEQ ID NO: WTYSPNFKTP ASTE D GRKKF ADTSLKLMKD4: bold letters are LGFDGIDIDW EYPEDEKQAN DFVLLLKACRsubstituted amino acids EALDAYSAKH PNGK R FLLTI ASP V GPQNYNKLKLAEMDKY LDFWNLMAYD FSGSWDKVSG HMSNVFPSTT KPESTPFSSD KAVKDYIKAGVPANKIVLGM PLYGRAFAST DGI M TSFNGV 8 Modified Cts1₁₀₅₋₃₁₀ 98%VYGCIK QMYLLKKNNR NLKTLLSIGG identical to SEQ ID NO:WTYSPNFKTP ASTEEGRKKF  I DTSLKLMKD 4; bold letters areLGFDGIDIDW EYPEDEKQAN DFVLLLKACR substituted amino acids D ALDAYSAKH PNV KKFLLTI ASPAGPQNYN KLKLAEMDKY LDFWNLMAYD FSGSWDKVSGHMSNVFPSTT KPESTPFSSD KAVKDYIKAG VPANKIVLGM PLY L RAFAST DGIGTSFNGV 9Modified Cts1₁₀₅₋₃₁₀ 95% VYGCIK QMYLL H KNNR NLKTLLSI VLidentical to SEQ ID NO: WTYSPNFKTP  I STEEGRKKF ADTSLKLMKD4; bold letters are LGFDGIDIDW EYP D DEKQAN DFVLLLKACRsubstituted amino acids EALDAYSAKH PNGK R FLLTI ASPAGPQNYN KL RLAEMDKY LDFWNLMAYD FSGSWDKVSG HMSNVFPSTT KPESTPFSS E  KAVKDYIKAGVPANKIVLGM PLYG H AFAST  E GIGTSFNGV 10 Modified Cts1₁₀₅₋₃₁₀ 95% V WGCIK QMYLLKKNNR NLKTLLSIGG identical to SEQ ID NO: WTYSPNFK NP ASTEEGRKKF ADTSLKLMKD 4; bold letters are LGFDGIDIDW EYPE E EKQAN DFVM LLKACR substituted amino acids EALDAYSVKH PNGKK Y LLTI ASPAGPQNYNKLKLAEMDKY LDFWNLMAYD FSGSWDKVSG HMS Q VFPSTT KPEST I FSSD KAVKDYI R AGVPANKIVL A M PLYGRAFAST DGIGTSFNGV 11 Modified Cts1₁₀₅₋₃₁₀ 90% VYG NIK QMYLL R KNNR NLKTLLSIGG identical to SEQ ID NO: WTYSPNFKT A  AS NEEGRKKF ADTSL H LMKD 4: bold letters are LGF EA IDIDW EYP DED KQAN D WVLLL H ACR substituted amino acids EALDAYSAKH PNGK R FLLTI ASPAGPQNYNKLKLAEMDKY LDFWNLMAYD FSGSWDKVS V HMSNVFPSTT  R PESTPF T SD KAVKDYIKAGVPAN RV VLGM PLYGR L FAST DGIGT T FNGV 12 Modified Cts1₁₀₅₋₃₁₀ 90% VYGCIR  QMYLLK H NNR NLKTL V SIGG identical to SEQ ID NO: WTYSPNF RTP ASTEEGRKKF ADTSLKLMKD 4; bold letters are LGFDGIDIDW EYP DEEKQAN DFVLL V KAC K substituted amino acids E V LDAYSAKH PNG HRFLLTI ASPAGPQNYN KLKLAEMDKY LDFWNLM I YD FSGSWDKV T G HMSNVFPSTT  RPESTPF T SD KAVK E YIKAG VP L NKIVLGM PLY A RAFAST DGI V TSFN M V

In some embodiments, the Cts1 peptide has a sequence that is 100%identical to SEQ ID NO: 4. In some embodiments, the Cts1 peptide has asequence that is 99% identical to SEQ ID NO: 4. In some embodiments, theCts1 peptide has a sequence that is 98% identical to SEQ ID NO: 4. Insome embodiments, the Cts1 peptide has a sequence that is 97% identicalto SEQ ID NO: 4. In some embodiments, the Cts1 peptide has a sequencethat is 96% identical to SEQ ID NO: 4. In some embodiments, the Cts1peptide has a sequence that is 95% identical to SEQ ID NO: 4. In someembodiments, the Cts1 peptide has a sequence that is 90% identical toSEQ ID NO: 4. In some embodiments, the Cts1 peptide has a sequence thatis 85% identical to SEQ ID NO: 4. In some embodiments, the Cts1 peptidehas a sequence that is 80% identical to SEQ ID NO: 4. In someembodiments, the Cts1 peptide has a sequence that is 75% identical toSEQ ID NO: 4. In some embodiments, the Cts1 peptide has a sequence thatis 70% identical to SEQ ID NO: 4. Non-limiting examples of peptides thatare similar to SEQ ID NO: 4 are shown in Table 1.

In other embodiments, the present invention also features peptides thatare similar to SEQ ID NO: 4, e.g., peptides wherein one amino acid isdifferent, two amino acids are different, three amino acids aredifferent, four amino acids are different, five amino acids aredifferent, six amino acids are different, seven amino acids aredifferent, eight amino acids are different, nine amino acids aredifferent, ten amino acids are different, more than 10 amino acids aredifferent, more than 20 amino acids are different, more than 30 aminoacids are different, 20-30 amino acids are different, 1-10 amino acidsare different, 10-20 amino acids are different, 30-40 amino acids aredifferent, 40-50 amino acids are different, etc. Non-limiting examplesof peptides that are similar to SEQ ID NO: 4 are shown in Table 1.

In some embodiments, the present invention provides an isolated peptideaccording to SEQ ID NO: 3. The present invention also feature peptidesthat are similar to SEQ ID NO: 3, e.g., peptides wherein one amino acidis different, two amino acids are different, three amino acids aredifferent, four amino acids are different, five amino acids aredifferent, six amino acids are different, seven amino acids aredifferent, eight amino acids are different, nine amino acids aredifferent, ten amino acids are different, more than 10 amino acids aredifferent, more than 20 amino acids are different, more than 30 aminoacids are different, 20-30 amino acids are different, 1-10 amino acidsare different, 10-20 amino acids are different, 30-40 amino acids aredifferent, 40-50 amino acids are different, etc. Stated differently, insome embodiments, the peptide is at least 75% identical to SEQ ID NO: 3.In some embodiments, the peptide is at least 80% identical to SEQ ID NO:3. In some embodiments, the peptide is at least 85% identical to SEQ IDNO: 3. In some embodiments, the peptide is at least 90% identical to SEQID NO: 3. In some embodiments, the peptide is at least 95% identical toSEQ ID NO: 3. In some embodiments, the peptide is at least 96% identicalto SEQ ID NO: 3. In some embodiments, the peptide is at least 97%identical to SEQ ID NO: 3. In some embodiments, the peptide is at least98% identical to SEQ ID NO: 3. In some embodiments, the peptide is atleast 99% identical to SEQ ID NO: 3. In some embodiments, the peptide isat least 100% identical to SEQ ID NO: 3.

In some embodiments, the isolated Cts1 peptide has a sequence accordingto SEQ ID NO: 5. In some embodiments, the isolated Cts1 peptide has asequence according to SEQ ID NO: 6. In some embodiments, the isolatedCts1 peptide has a sequence according to SEQ ID NO: 7. In someembodiments, the isolated Cts1 peptide has a sequence according to SEQID NO: 8. In some embodiments, the isolated Cts1 peptide has a sequenceaccording to SEQ ID NO: 9. In some embodiments, the isolated Cts1peptide has a sequence according to SEQ ID NO: 10. In some embodiments,the isolated Cts1 peptide has a sequence according to SEQ ID NO: 11. Insome embodiments, the isolated Cts1 peptide has a sequence according toSEQ ID NO: 12.

In some embodiments, the Cts1 peptide having a sequence of theabove-mentioned mentioned sequences has a sequence that is at least 100%identical to the aforementioned sequences. In some embodiments, the Cts1peptide having a sequence of the above-mentioned sequences has asequence that is at least 99% identical to the aforementioned sequences.In some embodiments, the Cts1 peptide having a sequence of theabove-mentioned sequences has a sequence that is at least 98% identicalto the aforementioned sequences. In some embodiments, the Cts1 peptidehaving a sequence of the above-mentioned sequences has a sequence thatis at least 97% identical to the aforementioned sequences. In someembodiments, the Cts1 peptide having a sequence of the above-mentionedsequences has a sequence that is at least 96% identical to theaforementioned sequences. In some embodiments, the Cts1 peptide having asequence of the above-mentioned sequences has a sequence that is atleast 95% identical to the aforementioned sequences. In someembodiments, the Cts1 peptide having a sequence of the above-mentionedsequences has a sequence that is at least 90% identical to theaforementioned sequences. In some embodiments, the Cts1 peptide having asequence of the above-mentioned sequences has a sequence that is atleast 85% identical to the aforementioned sequences. In someembodiments, the Cts1 peptide having a sequence of the above-mentionedsequences has a sequence that is at least 80% identical to theaforementioned sequences. In some embodiments, the Cts1 peptide ofhaving a sequence of the above-mentioned sequences has a sequence thatis at least 75% identical to the aforementioned sequences.

The present invention may also feature peptides that are similar to theaforementioned sequences, e.g., peptides wherein one amino acid isdifferent, two amino acids are different, three amino acids aredifferent, four amino acids are different, five amino acids aredifferent, six amino acids are different, seven amino acids aredifferent, eight amino acids are different, nine amino acids aredifferent, ten amino acids are different, more than 10 amino acids aredifferent, more than 20 amino acids are different, more than 30 aminoacids are different, 20-30 amino acids are different, 1-10 amino acidsare different, 10-20 amino acids are different, 30-40 amino acids aredifferent, 40-50 amino acids are different, etc.

The present invention also features nucleic acids that encode any of thepeptides disclosed herein (e.g., nucleic acids that encode SEQ ID NO: 3,SEQ ID NO: 4, a peptide similar thereto, etc.).

The present invention also provides expression vectors that can produceany of the peptides disclosed herein.

The present invention also provides peptide constructs comprising one ofthe peptides disclosed herein (e.g., SEQ ID NO: 4, SEQ ID NO: 3, etc.)attached to or linked (directly or indirectly) to a component used tobind the peptide to a solid support. Methods and reagents used forlinking or binding a peptide to a solid support are well known to one ofordinary skill in the art.

In some embodiments, the Cts1 peptide is attached to a solid support. Insome embodiments, the Cts1 peptide is covalently bound to a solidsupport. In other embodiments, the Cts1 peptide is non-covalently boundto a solid support. In some embodiments, a solid support may include butis not limited to solid surface, plastic, or streptavidin-coated plate.

In some embodiments, the Cts1 peptide is covalently attached to acomponent. In some embodiments, the Cts1 peptide is covalently bound toa component. In some embodiments, the Cts1 peptide is non-covalentlyattached to a component. In some embodiments, the Cts1 peptide isnon-covalently bound to a component. In some embodiments, the componentattaches the Cts1 peptide to a peptide that is on a solid support.Non-limiting examples of the component may include but are not limitedto biotin, a biotin mimic (SEQ ID NO: 27), a linker (SEQ ID NO: 26) or apeptide sequence that comprises SEQ ID NO: 26 followed by SEQ ID NO: 27.In other embodiments, a component may include additional amino acidsequences that bind to streptavidin like biotin does.

In some embodiments, the isolated Cts1 peptide is covalently attached toa component, wherein the component attaches to a peptide that is on asolid support.

In some embodiments, the isolated Cts1 peptide maintains itsconformational shape. In other embodiments, the isolated Cts1 peptidemaintains its conformational shape while attached to a component. Inother embodiments, the isolated Cts1 peptide maintains itsconformational shape while covalently attached to a component. In otherembodiments, the isolated Cts1 peptide maintains its conformationalshape while non-covalently attached to a component. In some embodiments,the isolated Cts1 peptide maintains its conformational shape while boundto a component. In other embodiments, the isolated Cts1 peptidemaintains its conformational shape while covalently bound to acomponent. In other embodiments, the isolated Cts1 peptide maintains itsconformational shape while non-covalently bound to a component.

The present invention may feature an assay platform for detectinganti-coccidioidal antibodies. In some embodiments, the platformcomprises a solid support. In other embodiments the platform comprisesan isolated Cts1 peptide having a sequence according to SEQ ID NO: 4 andcontaining at least one substitution modification relative to SEQ ID NO:4, wherein the Cts1 peptide is attached to the solid support.

In some embodiments, the isolated Cts1 peptide has a sequence accordingto SEQ ID NO: 5. In some embodiments, the isolated Cts1 peptide has asequence according to SEQ ID NO: 6. In some embodiments, the isolatedCts1 peptide has a sequence according to SEQ ID NO: 7. In someembodiments, the isolated Cts1 peptide has a sequence according to SEQID NO: 8. In some embodiments, the isolated Cts1 peptide has a sequenceaccording to SEQ ID NO: 9. In some embodiments, the isolated Cts1peptide has a sequence according to SEQ ID NO: 10. In some embodiments,the isolated Cts1 peptide has a sequence according to SEQ ID NO: 11. Insome embodiments, the isolated Cts1 peptide has a sequence according toSEQ ID NO: 12.

Without wishing to limit the invention to any theory or mechanism, it isbelieved that the substitution modification helps improve sensitivityand/or helps eliminate cross reactivity.

In some embodiments, the assay platform is for an ELISA assay. In someembodiments, the platform is for a complement fixation assay.

In some embodiments, the solid support is a well. In some embodiments,the well is a part of a microwell plate. In some embodiments, the solidsupport is a microwell. In other embodiments, the solid support is amicrowell plate. In other embodiments, the solid support is a solidsurface. In some embodiments, the solid support is plastic. In someembodiments, the solid support is a streptavidin-coated plate.

In some embodiments, the isolated Cts1 peptide is covalently attached toa component. In some embodiments, the component attaches to a peptidethat is on a solid support. In some embodiments, the isolated Cts1peptide maintains its conformational shape. In other embodiments, theisolated Cts1 peptide maintains its conformational shape while attachedto the platform. In some embodiments, the isolated Cts1 peptidemaintains its conformational shape while attached to a solid support.Non-limiting examples of the component may include but are not limitedto biotin, a biotin mimic (SEQ ID NO: 27), a linker (SEQ ID NO: 26) or apeptide sequence that comprises SEQ ID NO: 26 followed by SEQ ID NO: 27.

Additionally, the present invention may feature an isolated Cts1 peptidehaving a sequence that is at least 90% identical to SEQ ID NO: 4,wherein the Cts1 peptide is covalently attached to a component, whereinthe component attaches to a peptide that is coated onto a solid support.In some embodiments, the isolated Cts1 peptide maintains itsconformational shape.

The present invention also provides assay platforms for detectinganti-coccidioidal antibodies. For example, the assay platform maycomprise a surface, such as a well, wherein a peptide of the presentinvention (e.g., SEQ ID NO: 4) is bound to the surface. A sample may beintroduced to the assay platform, wherein the sample is contacted withthe peptide bound to the surface. Assays such as this may be similar toan ELISA.

In some embodiments, the surface the peptide is bound to may include butis not limited to a solid support, a solid surface, plastic, orstreptavidin-coated plate. In other embodiments, the peptide may beattached to the surface by, but not limited to, biotin, a biotin mimic(SEQ ID NO: 27), a linker (SEQ ID NO: 26) or a peptide sequence thatcomprises SEQ ID NO: 26 followed by SEQ ID NO: 27. In other embodiments,the peptide may be attached to the surface by other amino acid sequencesthat bind to streptavidin like biotin does.

EXAMPLE

The following is a non-limiting example of the present invention. It isto be understood that said example is not intended to limit the presentinvention in any way. Equivalents or substitutes are within the scope ofthe present invention.

Methods:

Human sera. A serum library composed of remnant specimens tested foranti-coccidioidal antibodies by immunodiffusion at the Southern ArizonaVeterans Health Care System Medical Center was used for these studies.All specimens were de-identified, and the University of ArizonaInstitutional Review Board has determined that their use was not humanexperimentation. For some studies, a serum pool was created fromportions of 50 separate sera. The calculated geometric mean CF titer forthe sum of the individual sera was 1:12, and when the pool was testeddirectly by quantitative immunodiffusion, the titer was 1.8.

Recombinant antigen preparation. Full-length (FL) CTS1 was produced byFOR from a cDNA library (Dugger K O, Villareal K M, Nguyen A, ZimmermannC R, Law J H, Galgiani J N. Cloning and sequence analysis of the cDNAfor a protein from Coccidioides posadasii with immunogenic potential.BiochemBiophysResCommun 1996; 218(2): 485-9) using primers shown inTable 2. Sequence for this amplimer matched that submitted to Genbank(U60807) (Zimmermann C R, Johnson S M, Martens G W, White A G,Pappagianis D. Cloning and expression of the complement fixationantigen-chitinase of Coccidioides immitis. Infect Immun 1996; 64(12):4967-75). Truncations of and modifications of rCTS1 were prepared by PCRusing primers also shown in Table 2.

TABLE 2 Primers used to produce rCTS1 and its truncations. SEQ Amino ID# Name Acid DNA Sequence NO.: FL CF-1 1-*** GGATCCCCGAATTCATGAGGTTCCTT13 ATTGGCTTTACTTACTTACTCTC FL CF-427 ***-427 CGGGATCCATGATGATGATGATGATG14 ACTTGGCATCCCATTCTTCTTGAG 1 20-Fw 20-*** TACTTCCAATCCAATGCGATGTCAAG 15TATGCCCAATTATTATCCAG 2a 111-Fw 111-** TACTTCCAATCCAATGCGCAAATGTA 16CTTGCTCAAGAAGAACAACCGGAAC 3 310-Rv ***-310 TTATCCACTTCCAATGCGCTAAACAC 17CGTTGAAGCTAGTGCCGATCCCATC 3a 310_BMP_ ***-310 TTATCCACTTCCAATGCGCTACCACC18 Rv GAACTGCGGGTGACGCCAAGCGGAG CTGGCGCTGGCGCCGCCGCCAACAC CGTTGAAGCTAGT2b Frag1_ 105-*** TACTTCCAATCCAATGCGGTTTACGG 19 105_FwCTGTATCAAGCAAATGTACTTGCTCA AGAAG 4 Frag1_ ***-164TTATCCACTTCCAATGCGCTAATCAAA 20 164_Rv GCCAAGGTCCTTCATCAACTTCAGAG ATGTG 5Frag2_ 154--** TACTTCCAATCCAATGCGTCTCTGAA 21 154_FwGTTGATGAAGGACCTTGGCTTTGATG G 6 Frag2_ ***-212 TTATCCACTTCCAATGCGCTATGAAG22 212_Rv CAATAGTGAGCAAGAATTTCTTGCCA TTCGGG 7 Frag3_ 202-***TACTTCCAATCCAATGCGAATGGCAA 23 202_Fw GAAATTCTTGCTCACTATTGCTTCACC GG 8Frag3_ ***-252 TTATCCACTTCCAATGCGCTACATGT 24 252_RvGGCCAGACACTTTGTCCCAGCTG 9 Frag4_ 240-*** TACTTCCAATCCAATGCGGACTTCAG 25240_Fw CGGCAGCTGGGACAAAGTG

For initial studies with rCTS1₂₀₋₃₁₀ (SEQ ID NO: 2) and rCTS1₁₁₁₋₃₁₀(SEQ ID NO: 3) were expressed in Escherichia coil BL21 DE3 gold andextracted with 8M urea buffers and disrupted by ultrasound treatment atroom temperature for 1 hour. The purification was accomplished byaffinity binding to nickel-NTA column. After collecting and poolingeluate fractions containing the expressed peptide as judged by SDS-PAGEand immunoblot, the pool was placed in dialysis tubing, and the urea wasslowly removed by making twice daily 1-liter changes of the renaturationbuffer (150 mM of Sodium Chloride (NaCl), 1 mM ofEthylenediaminetetraacetic acid (EDTA), 5 mM of Glutathione reduced(GSH), 0.5 mM of Glutathione oxidized (GSSG), 20 mM of Tris pH9.5 and10% of Glycerol). The stepwise gradient of urea was 6.5, 5, 3.5, 1, and0 M. Following renaturation (e.g., dialyze to PBS), gel-filtration wasconducted with a P100 fine (Bio-Rad) 50 ml packed in an adaptor column2.0+20 cm at a flow rate of 0.1 ml/min, Proteins were analyzed by PAGEand subsequent immunoblotting, for example, FIG. 1B shows SDS PAGE of E.coli-expressed rCTS1 truncations after Ni-NTA affinity purification.FIG. 1A shows an immunoblot of E. coli-expressed rCTS1 truncations withCF+ human serum.

For subsequent studies, amplimers were cloned into Ligation Independent

Cloning Vectors pMCSG7 that contained a T7 promoter and N-terminalHis-tag, as described Eschenfeldt et al 2009 (Eschenfeldt W H, Lucy 5,Millard C S, Joachimiak A, Mark I D. A family of LIC vectors forhigh-throughput cloning and purification of proteins. Methods Mol Biol2009; 498; 105-15) and confirmed by sequencing. The rCTS1₁₀₅₋₃₁₀truncation was soluble upon E. coli lysis, and thus, did not need to berefolded as a step of collection. Further, it gave significantly higheryield per gram of bacterial pellet than the rCTS1₁₀₅₋₃₁₀ truncation.Thus, the rCTS1₁₀₅₋₃₁₀ truncation was used in place of the rCTS1₁₁₁₋₃₁₀truncation. An additional construct of rCTS1₁₀₅₋₃₁₀ involved creating abiotin mimetic protein tag at the C-terminal end of the aa105-310truncation by adding a linker region GGGASAS (SEQ ID NO: 26) followed bythe decapeptide, SAWRHPQFGG (SEQ ID NO: 27) via PCR.

Vectors for the various rCTS1 constructs were cloned into Escherichiacoli BL21 DE3 gold cells and purified as in Neubert et al. 2017 (NeubertM J, Dahlmann E A, Ambrose A, Johnson M D L. Copper Chaperone CupA andZinc Control CopY Regulation of the Pneumococcal cop Operon. mSphere2017; 2(5)) with modifications. After initial purification usingimmobilized nickel-affinity chromatography (IMAC) (HisTrap FF, GEHealthcare), protein was further purified by size-exclusionchromatography (SEC) (Superdex 200, GE Healthcare) using a buffer of 20mM Tris pH 8, 200 mM NaCl, and 5% glycerol. Additionally, using theprimers shown in Table 2, four fragments were made of the aa105-310truncation: Fragment 1 (aa105-164), Fragment 2 (aa154-212), Fragment 3(aa202-252), and Fragment 4 (aa240-310). Based on either no peak orobservable band on a gel from the nickel elution, fragments #1, 3, and 4were determined to be insoluble. To resolve this, truncations for thesefragments were cloned into pMCSG9 that contained a maltose bindingprotein (MBP) linker to increase solubility, and the growth andpurification process repeated. SEC peaks containing pure rCTS1truncation fragments (as determined by SDS-PAGE), with (fragments #1, 3,4) or without (fragment #2) MBP were concentrated if necessary (i.e.Amicon® Ultra Centrifugal Filters with molecular weight cutoffs) andconcentration was determined by absorbance at 280 nm using molecularweight and extinction coefficient. Samples at >10 μM were aliquoted intothin-walled FOR tubes, and flash-frozen using liquid N₂

Immunoblot analysis. All expression products were analyzed on standardSDS PAGE. Because truncations differed in size, peptides were calculatedin molarity and approximately 200 um/lane of each soluble peptide wasloaded equally on 4-20% Ready GEL (Bio-Rad, Cat #4561093), andsubsequently the gels were stained with Coomassie. Separated proteinswere transferred from the gel to a nitrocellulose membrane, rinsed withTris-buffered saline containing 0.05% Tween-20 (TBST) before blockingwith 5% normal goat sera (NGS) in TBS-T for either one hour at roomtemperature or overnight at 4° C. Two identical immunoblots wereperformed, one with sera from the serum bank (CF=1:128) and the otherwith normal human serum. The sera were diluted 1:1000. Immunoblots werethen washed, stained with alkaline phosphatase-conjugated goatanti-human IgG antibody, washed again, and immunoreactive bandsvisualized with colorimetric alkaline phosphatase substrate reagents.

As indicated in the results, for one competition study, a truncation(rCTS1₁₁₁₋₃₁₀) was mixed with the CF+ sera at 1 mM or 10 mM (5×or 50×theamount that was loaded to the gel) prior to application to the membraneand resulting binding to rCTS1(aa20-310) was assessed.

Immunoblots demonstrated that both the a.a. 20-310 (Cts1₂₀₋₃₁₀; SEQ IDNO: 2) and the a.a. 111-310 (Cts1₁₁₁₋₃₁₀; SEQ ID NO: 3) recombinantpeptides reacted to sera from patients with coccidioidomycosis thatcontained CF antibodies but did not react with sera from uninfectedpatients. Absorption of CF positive sera with a.a. 111-310 (Cts1₁₁₁₋₃₁₀;SEQ ID NO: 3) eliminated immunoblot binding of the sera to a.a. 20-310(Cts1₂₀₋₃₁₀; SEQ ID NO; 2) (data not shown).

ELISA. Initial studies were carried out by coating standard plastic96-well microplates (Thermo Scientific, Cat, # 80040E0910) with 100ng/well of recombinant peptides at 4° C. for overnight. Preliminarystudies demonstrated that his amount of peptide per well was notrate-limiting. After triplicate washing with PBST, coated wells wereblocked with 5% milk in PBST at room temperature for 30 min, excess wasremoved, and 100 μl of the serum pool, or individual human CF+ sera usedto prepare the pool were added to duplicate wells at various dilutions.Duplicate wells filled with PBS were also included. After incubation atroom temperature for 1 hour, wells were washed 3 times with PBST. Then,affinity-purified peroxidase-labeled goat anti-human IgG antibodies(KPL, Cat. #474-1006, diluted 1:10,000) was added and left at roomtemperature for 1 h, after which the wells were washed 3 times withPBST. Finally,

SuperBlue TMB Microwell Peroxidase Substrate (KPI_(—) Cat. #52-00-02)was added and 10 min later 1N HCl stop solution is added. Opticaldensities (OD) were read at 450 nm, Same-day standard ELISA curves wereproduced by coating wells with goat anti-human antibody (IgG) or PBS andincubated with human immunoglobulin at concentrations from 2-14 ng/ml.Experimental OD measurements minus background were plotted on thestandard curves within the OD range of 0.10 to 0.80, multiplied by theserum dilution factor and expressed as μg/ml of IgG.

For later studies, Pierce Streptavidin coated high binding capacity96-well plates (Thermo Scientific, Cat. #1550) were used for studieswith the biotin mimic-tagged peptides. The rest of the procedure wasformed identically to that described above except that tris-buffersaline, 0.1% BSA, 0.05% Tween-20 were used as the wash buffer instead ofPBST, and the standard curve was constructed with whole molecule biotinconjugated to human IgG (Rockland, Cat, #009-0602) at concentrations of2.5 to 10.0 ηg/ml.

Statistical Analysis. The correlation of CF antibody titers with ELISAresults was estimated as the significance of the slope of the linearregression paired results as being non-zero.

Anti-coccidioidal antibody binding is restricted to rCTS1₁₁₁₋₃₁₀, butnot to smaller rCTS truncations.

Initial studies focused on the rCTS1₁₁₁₋₃₁₀ and compared it torCTS1_(20-310.) As shown in FIGS. 1A-1B, the shorter truncation (27.4kDa) demonstrates at least as much binding as the previously publishedrCTS1₂₀₋₃₁₀ (34.7 kDa). Further, adding increasing concentrations of_(rCTS)1₁₁₋₃₁₀ to antisera used to perform immunoblotting of rCTS1₂₀₋₃₁₀virtually blocked all antibody binding to the membrane-bound peptide.This indicates that antibody binding to rCTS1₁₁₁₋₃₁₀ accounted fornearly all of the binding to rCTS1₂₀₋₃₁₀ (FIG. 4).

Next, four overlapping truncations (rCTS1₁₀₅₋₁₆₄, rCTS1₁₅₄₋₂₁₂,rCTS1₂₀₂₋₂₅₂, and rCTS1₂₄₀₋₃₁₀) were produced as shown in FIGS. 1A-1B.Each truncation overlapped by at least 11 residues. For these and futurestudies, rCTS1₁₀₅₋₃₁₀ (SEQ ID NO: 4; Table 1) was substituted forrCTS1₁₁₁₋₃₁₀ because this slightly larger truncation produced aseveral-fold greater yield of expression product and did not requiredenaturation and refolding to remain soluble. Of these truncations, onlysubunit #2 was soluble without MBP, and only subunit #3 (rCTS1₂₀₂₋₂₅₂)was soluble when the MBP was cleaved from the expression product. Of thefour soluble truncations, western blots failed to show any significantbinding to any of the subunits (FIGS. 5A & 5B). Also, comparing ELISAresults when rCTS1₁₀₅₋₃₁₀ (SEQ ID NO: 4) or each of the four subunitpeptides, or MBP alone, found virtually no binding by CF positive serum(FIG. 2). These unexpected findings suggested that most if not all ofthe binding of serum antibodies to rCTS1₁₀₅₋₃₁₀ was directed atconformational or discontinuous epitopes rather than epitopes of aprimary amino acid sequence. Without wishing to limit the presentinvention to any theory or mechanism, it is believed that a similarassay may be used as a diagnostic test procedure.

Sensitivity and reproducibility of antibody binding to rCTS₁₀₅₋₃₁₀ isimproved by tag-binding the peptide to the ELISA well.

Preliminary quantitative ELISA studies were performed using rCTS1₁₀₅₋₃₁₀absorbed directly to plastic microtiter wells. However, day to dayreplication of binding of a pool created from CF antibody positivepatient sera (CF titer of the pool was 1:8) resulted in unsatisfactoryvariability. Because the epitope mapping suggested that conformation ofthe peptide was critical, it was postulated that adhering the peptidedirectly to the plastic might result in steric distortion in anuncontrolled manner. Moreover, anchoring the antigen uniformly to thewell by means of a terminal tag might result in the anchored antigenretaining its fluid phase conformation such as is the case in bothclassic CF antibody and immunodiffusion assays. To study this, an aminoacid sequence mimic of biotin, SAWRHPQFGG (SEQ ID NO: 27), was cloned tothe C-terminal of rCTS1₁₀₅₋₃₁₀. The biotin mimic sequence readily bindsto streptavidin-coated plates to low μM affinity. Using thisreconfigured assay, the quantitative ELISA results of replicates on 12separate days ranged from 92 μg/ml to 174 μg/ml (average=112 μg/ml,sem±25).

The quantitative antibody detection by ELISA performed was compared withrCTS1₁₀₅₋₃₁₀ absorbed directly to the well of uncoated plastic platesand with the biotin-mimic tag bound to streptavidin coated plates. Asshown in Table 5, results with the mimic-tagged peptide uniformlydetected more antibodies than did the peptide absorbed directly to theplastic with detection ratios ranging from 19- to 193-fold greater.Using the tag-bound rCTS₁₀₅₋₃₁₀ peptide, the 50 sera used to constructthe serum pool individually were assayed, and the results are shown inFIG. 3. There is a significant relationship between the ELISA-measuredanti-rCTS1₁₀₅₋₃₁₀ IgG concentration and the CF titer (p=0.0085) althoughthe r² is only 0.14.

TABLE 3 Difference of results between quantitative ELISA results from 11individual human sera using different methods of binding the antigen tothe well of the plates. Antibody binding (ug/ml) to rCTS1₁₀₅₋₃₁₀ IDCFProtein directly Protein with biotin mimic bound Ratio of biotin literto the plastic to Streptavidin-coated wells mimic:plastic 2 0.096 14.00146 2 0.491 12.80 26 2 0.023 4.45 193 2 1.437 78.75 55 4 0.454 8.50 19 41.525 55.38 36 4 0.149 16.45 110 8 0.566 34.69 61 8 0.218 6.95 33 161.028 24.60 24 16 0.08 14.40 180 Average fold difference 79

The present absorption studies where rCTS1₁₁₁₋₃₁₀ was mixed withCF-positive serum virtually eliminated any binding to rCTS1₂₀₋₃₁₀ on thenitrocellulose membrane. However, none of four smaller, overlappingtruncations of this region showed any appreciable antibody binding. Thesimplest explanation for this unexpected finding is that antibodybinding to Coccidioides-specific epitope(s) depends upon conformation(s)present CTS1₁₁₁₋₃₁₀ that is lost with the individual smaller peptides.The expression of rCTS1₁₀₅₋₃₁₁ was found to be achieved in abundantquantities for serologic purposes.

Although the exact structure of the Coccidioides-specific conformationalepitopes has not been identified, the present findings do suggest apossible reason why a recombinant CTS1 has not yet been used to developan ELISA to mimic the quantitative CF antibody assay. An ELISAconfiguration with rCTS1 absorbed directly to the plastic well mightwell distort the protein's conformation in unpredictable ways and reduceits antibody binding. As found to be the case for CTS1₁₀₅₋₃₁₀ whencompared to results with an ELISA where the peptide used a biotin-mimictag sequence for oriented binding to streptavidin-coated wells (Table3). Thus, using a biotin-mimic tag may offer a technical modification toimprove a quantitative ELISA.

A general correlation between CF titer and ug/ml of antibody binding toCTS1₁₀₅₋₃₁₀ was observed (FIG. 3). The use of a standard curve toquantitate antibody binding has the advantage of allowing results fromsequential specimens over time to be compared without running twospecimens at the same time as has been recommended for optimal CFantibody titer comparisons. The correlation not being better than whatwas observed could be due to several factors. ELISA measures antibodybinding to monomeric antigens which is influenced by the distribution ofantibody avidities in different patient sera whereas CF antibody assaysare based upon antigen-specific immune-complex formation. Therelationship of these two dependencies is likely not uniform indifferent patients' sera. It is possible that better agreement betweenELISA and CF results might be achieved using different conditions forantigen-antibody binding than those that were reused in the presentstudies. For example, if low-avidity antibodies in some specimensvariably contributed to ELISA results, then adding urea might providemore stringency for antibodies binding to the plate and improveagreement.

The CF antibody assay titers have prognostic importance that wasdescribed in the 1950s. It should be noted that those pioneering studieswere conducted by a single research laboratory, focused upon CM andbefore there were any effective therapies for this disease. Currently,CF antibody tests are provided for patient care by several referencelaboratories. That antifungal therapy might alter the prognosticrelationships noted earlier has recently been raised. The difference inindividual sera between the present quantitative ELISA and CF antibodytiters provides an opportunity in future studies to determine whetherone or the other provide more precise prognostic information toclinicians.

Even if a quantitative ELISA is no more useful than CF antibody testresults, it offers several practical advantages. ELISA is less complexthan the CF test. ELISA reagents are readily available, and the formatis familiar to most clinical laboratories. Also, ELISA is less laborintensive and can be automated. For all of these reasons, the presentstudies reported herein will provide further incentive for thedevelopment of a standardized quantitative ELISA as an alternative tothe CF antibody assay.

The present invention describes isolated peptides of Cts1 that retainaffinity for CF antibodies. These results help provide the basis for areference enzyme-linked immunoassay to mimic quantitative resultscurrently produced by the originally described CF antibody detectionassay. FIG. 6 demonstrates that absorbance resulting from enzymeconjugate detection of antibodies is proportional to the CF titers.Therefore, measurement of absorbance of several different dilutions of apatient serum or other bodily fluid will result in absorbance curvesthat are proportional to the amount of CF antibody activity in thespecimen. Using specimens of known CF antibody titers, a final protocolcould determine the absorbance threshold at which the CF titer wouldcorrespond to a specific specimen dilution.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

As used herein, the term “about” refers to plus or minus 10% of thereferenced number.

Although there has been shown and described the preferred embodiment ofthe present invention, it will be readily apparent to those skilled inthe art that modifications may be made thereto which do not exceed thescope of the appended claims. Therefore, the scope of the invention isonly to be limited by the following claims. In some embodiments, thefigures presented in this patent application are drawn to scale,including the angles, ratios of dimensions, etc. In some embodiments,the figures are representative only and the claims are not limited bythe dimensions of the figures. In some embodiments, descriptions of theinventions described herein using the phrase “comprising” includesembodiments that could be described as “consisting essentially of” or“consisting of”, and as such the written description requirement forclaiming one or more embodiments of the present invention using thephrase “consisting essentially of” or “consisting of” is met.

What is claimed is:
 1. An isolated Cts1 peptide having a sequence thatis at least 90% identical to SEQ ID NO: 4 and containing at least onesubstitution modification relative to SEQ ID NO:
 4. 2. The isolated Cts1peptide of claim 1, wherein the Cts1 peptide has a sequence that is atleast 95% identical to SEQ ID NO:
 4. 3. The isolated Cts1 peptide ofclaim 1, wherein the Cts1 peptide has a sequence that is at least 99%identical to SEQ ID NO:
 4. 4. The isolated Cts1 peptide of claim 1,wherein the Cts1 peptide has a sequence according to SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, or SEQ ID NO:
 12. 5. The isolated Cts1 peptide of claim 1,wherein the Cts1 peptide is attached to a solid support.
 6. The isolatedCts1 peptide of claim 1, wherein the isolated Cts1 peptide is atruncated peptide of SEQ ID NO:
 1. 7. The isolated Cts1 peptide of claim1, wherein the isolated Cts1 peptide is covalently attached to acomponent, wherein the component attaches to a peptide that is on asolid support.
 8. The isolated Cts1 peptide of claim 7, wherein theisolated Cts1 peptide maintains its conformational shape.
 9. An assayplatform for detecting anti-coccidioidal antibodies, said platformcomprising: a. a solid support; and b. an isolated Cts1 peptide having asequence according to SEQ ID NO: 4 and containing at least onesubstitution modification relative to SEQ ID NO: 4, wherein the Cts1 ispeptide attached to the solid support.
 10. The platform of claim 9,wherein the Cts1 peptide has a sequence according to SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, or SEQ ID NO:
 12. 11. The platform of claim 9, wherein theisolated Cts1 peptide is a truncated peptide of SEQ ID NO:
 1. 12. Theplatform of claim 9, wherein the assay platform is for ELISAs.
 13. Theplatform of claim 9, wherein the assay platform is for a complementfixation assay.
 14. The platform of claim 9, wherein the solid supportis a well.
 15. The platform of claim 13, wherein the well is part of amicrowell plate.
 16. The platform of claim 9, wherein the isolated Cts1peptide is covalently attached to a component, wherein the componentattaches to a peptide that is on a solid support.
 17. The platform ofclaim 15, wherein the isolated Cts1 peptide maintains its conformationalshape.
 18. An isolated Cts1 peptide having a sequence that is at least90% identical to SEQ ID NO: 4, wherein the Cts1 peptide is covalentlyattached to a component, wherein the component attaches to a peptidethat is coated onto a solid support.
 19. The isolated Cts1 peptide ofclaim 18, wherein the isolated Cts1 peptide maintains its conformationalshape.
 20. The isolated Cts1 peptide of claim 18, wherein the isolatedCts1 peptide is a truncated peptide of SEQ ID NO: 1.